Strict regulation of the receptor tyrosine kinase (RTK)/extracellular signal-regulated kinase (ERK) pathway is essential for maintaining balanced growth in multi-cellular organisms. Several negative regulators of the pathway have been identified which include Sprouty proteins. Mammalian cells express four Sprouty isoforms (Sprouty1-4) in an ERK-dependent manner. In this study, we have examined the molecular mechanisms by which Sprouty proteins elicit their inhibitory effects on the RTK/ERK pathway, with special focus on the co-operation among Sprouty isoforms. The four mammalian Sprouty isoforms interact with each other, most probably to form hetero- as well as homo-oligomers through their C-terminal domains. Sprouty1 specifically interacts with Grb2, whereas Sprouty4 interacts with Sos1. Although any of the Sprouty isoforms by itself inhibits the fibroblast growth factor-2 (FGF-2)-induced activation of the ERK pathway significantly, hetero-oligomers show a more pronounced inhibitory activity. The hetero-oligomer formed between Sprouty1 and Sprouty4 exhibits the most potent inhibitory effect on ERK activation through its highly effective ability to suppress the association of Grb2-Sos1 complex with FRS2. The cooperative interactions observed among Sprouty isoforms could represent an advanced system that functions to regulate strictly the activation state of the RTK/ERK pathway in mammalian cells.